As a system for separating the components contained in a sample and individually collecting those components, a preparative separation chromatograph is commonly known, in which the components are temporally separated by a column in a high-performance liquid chromatograph or similar apparatus, and each component is subsequently collected with a fraction collector (for example, see Patent Literatures 1 and 2).
The preparative separation chromatograph includes a separation unit having a liquid-sending pump and a column, a detector located behind the separation unit, a fraction collector, as well as a controller for controlling those devices. The sample components which have been eluted from the column in a temporally separated form are sequentially detected by the detector (e.g. an ultraviolet visible spectrophotometer) and introduced into the fraction collector in the subsequent stage. In the fraction collector, the internal passage is switched according to the command from the controller so that the target components are respectively collected into fraction containers, such as vials.
Many preparative separation chromatographs are capable of so-called “automatic fractionation”, i.e. the automatic collection of the components eluted from the column. In automatic fractionation, the controller locates the beginning and ending points of the elution of each component based on the output signal from the detector. It controls the fraction collector so as to initiate the collecting operation after a predetermined amount of time from the beginning of the elution of one component and discontinue the operation after the predetermined amount of time from the end of the elution of the component. The “predetermined amount of time” corresponds to the amount of time required for a component which has passed through the detector to reach the collecting section of the fraction collector, which is determined by the length of the passage from the detector to the fraction collector, the flow velocity of the mobile phase, and other factors.
Automatic fractionation is roughly divided into two types: the “level method”, which uses a threshold, and the “slope method”, which uses a rate of change. In the level method, the point in time where the output signal from the detector exceeds a threshold is located as the beginning point of a chromatogram peak (the beginning of the elution of a component), while the point in time where the signal falls below the threshold is located as the ending point of the peak (the end of the elution of the component). However, as in the case of the gradient elution method in which the composition of the mobile phase is changed with time, if the background level of the chromatogram changes with time, it is difficult to correctly locate the beginning and ending points of the elution of the component by the level method. Furthermore, locating those points will be totally impossible if the background level exceeds the threshold.
In the slope method, the output signal from the detector is acquired at predetermined time intervals, and the rate of change from the previously acquired output signal is calculated. The point in time where the magnitude of the rate of change becomes greater than a positive predetermined slope value is located as the beginning point of a chromatogram peak (i.e. the beginning of the elution of a component), while the point in time where the absolute value of the rate of change becomes smaller than an absolute value of a negative predetermined slope value is located as the ending point of the chromatogram peak (i.e. the end of the elution of the component). For example, if the positive slope value is 200 μV/sec and the negative slope value is −200 μV/sec, the point in time where the slope of the change in the signal intensity of the detector exceeds 200 μV/sec is located as the beginning point of the peak. After passing the peak top, the rate of change in the signal intensity turns negative. Subsequently, the point in time where the absolute value of the rate of change becomes smaller than the absolute value (200 μV/sec) of the negative slope value is located as the ending point of the peak. In this manner, according to the slope method, the beginning and ending points of the peak are located by examining the rate of change in the output signal, and not the absolute value of the signal. This method can be used even when the background level changes with time.